Project Summary Lung cancer among never-smokers is currently one of the top ten causes of cancer mortality worldwide, with rates more than doubling in the past three decades. Its prevalence in women is consistent with results from epidemiologic and mechanistic studies suggesting that estrogen contributes to lung carcinogenesis. Estrogen can either stimulate cell growth via estrogen receptor (ER)-mediated signaling or be metabolized to genotoxic and mutagenic derivatives; a process that has received little attention. This group is the first to demonstrate the presence of multiple estrogen metabolites in the human lung, including carcinogenic 4-hydroxyestrogen (4- OHE). Preliminary data indicate that the level of 4-OHE is higher in lung tumors vs. adjacent normal tissue from patients with non-small cell lung cancer (NSCLC). Mice deficient in Cyp1b1, the enzyme responsible for 4-OHE production, develop fewer Kras-driven lung adenomas than wild-type controls. However, the mechanism by which 4-OHE promotes lung carcinogenesis remains unknown. The hypothesis of the proposed studies is that 4-OHE, a product of CYP1B1, causes activation of ER signaling and mutation of oncogenes within the lung; processes that increase the risk of lung cancer. Thus, inhibition of CYP1B1 activity could reduce the risk of lung cancer among never-smokers. In Aim 1, immortalized normal human bronchial epithelial cells with variable levels of CYP1B1 and COMT, the rate-limiting estrogen conjugation enzyme, will be employed to assess the ability of 17beta-estradiol or 4-OHE to induce: mutations in genes that drive lung cancer (EGFR, KRAS, p53, ALK, HER2, BRAF, PI3KCA), genomic and nongenomic activities of the ERs, and cellular transformation (anchorage- independent growth and tumor formation in SCID mice). The ability of estrogen metabolites (4-OHE) and ER signaling to cooperate to promote lung tumors formation will be evaluated in Aim 2 in Cyp1b1+/+ or -/- mice carrying a doxycycline-inducible EGFR mutation, in the presence and absence of the ER antagonist fulvestrant. Changes in tumor burden over time will be monitored by CT scans and tumor stage, the profile of pulmonary estrogen metabolites and the phosphorylation status of EGFR and its downstream targets will be determined at necropsy. The estrogen metabolite profile of lung tissue from healthy donors will be established for the first time and compared to that of NSCLC patients (normal tissue) in Aim 3 to determine if pulmonary 4-OHE levels differ in normal tissue from patients and healthy controls and correlate with the activity of CYP1B1 and COMT. The relationship between 4-OHE levels and specific polymorphic variants of CYP1B1 and COMT will also be explored. Data from the proposed studies are anticipated to provide novel insight into the contribution of estrogen metabolism to lung tumorigenesis in never-smokers and reveal the potential utility of CYP1B1 and/or the ERs as molecular targets for the prevention of NSCLC. This promising strategy could ultimately lead to a dramatic reduction in the risk of lung cancer faced by never-smokers worldwide.